model1 was the longest ischemia duration and simplest operation among the fourmodels
The mice unilateral limb ischemia model, which mimics critical limb ischemia in humans, was induced in mice and treated with four different surgical methods (Supplementary Fig 1a). After limb ischemia surgery, four groups mice were observed from days 0 to 21.
Limb ischemia inevitably affects motor function, as shown in Supplementary Fig 1b, we found that the motor function recovery of model2 and model3 were significantly faster than model1 and model4 at day 14 and basically returned to normal.
To investigate the recovery of blood perfusion, the blood perfusion was measured by Laser Doppler perfusion imaging (Supplementary Fig 1c). The result showed the blood perfusion percentage of model1 was significantly lower than model2 and model3 at the days 0, 14 and 21, but there was no significant difference between model1 and model4, the result indicated that the recovery of blood perfusion of model1 and model4 were slower than model2 and model3.
The satellite cells are activated to differentiate and fuse into myotube to promote skeletal muscle regeneration when suffered from ischemic damage. Regenerated skeletal muscle presents as central nuclear fibers, while mature skeletal muscle presents as eccentric nuclear fibers. The result showed the regenerated muscle fibers ratio of model1 was significantly higher than model2 and model3 at days 7 and 21. No significantly difference was found between model1 and model4 (Supplementary Fig 1d).
Our result indicated that model1 and model4 had were longest ischemia duration. Compared with model4, model1 may be a better choice due to its simple operation. Taken together, model1 was used in subsequent experimental studies.
PC protect ischemic damaged skeletal muscle by reducing the oxidative stress and promoting regeneration
As shown in Fig. 1a, the unilateral limb ischemia mice were performed by model1 method. After surgery, mice were treated with H2O, 20 mg/kg PC or 1 mg/kg PC, respectively. The results showed that the recovery of limb motor function in each group was time-dependent, and returned to normal at day 21. And 20 mg/kg PC were significantly faster than that in H2O and 1 mg/kg PC (Fig. 1b). Analysis of tissue regeneration in ischemic gastrocnemius at days 7 and 21 revealed that 20 mg/kg PC treated mice contained a higher number of regenerating myofibers than that in H2O and 1 mg/kg PC (Fig. 1c). However, there were no evidently difference between H2O group and 1 mg/kg PC group neither in motor function recovery nor in muscle regeneration. In order to further evaluate the muscle regeneration, the expression of myogenin was been detected by immunofluorescence analysis. Compared with saline, the number of myogenin positive cells in 20 mg/kg PC group were significantly increased at day 7 (Fig. 1d). To investigate whether these changes were due to differences in oxidative stress, the MDA levels were further detected both in plasma and in ischemia muscles at days 7 and 21 (Fig. 1e). Compared with H2O, 20 mg/kg PC induction resulted in significantly lower MDA levels both in plasma and ischemic muscle at day 7. Compared with 1 mg/kg PC, 20 mg/kg PC induction resulted in significantly lower MDA plasma levels at day 7. Meanwhile, no significantly difference was found among three groups at day 21.
These results suggested that different concentrations of PC cause different effects on ischemic muscle, that is, 20 mg/kg PC can protect ischemia-induced muscle damage, which may be achieved by resisting oxidative stress and promoting ischemic muscle regeneration, H2O and 1 mg/kg PC have no such effect. Considering the regeneration of ischemic muscle was most obvious at day 7, so unilateral limb ischemia mice treated with 20 mg/kg PC for 7 days were chosen in the following study.
PC promote HSKMSCs myogenic differentiation in vitro under hypoxic-ischemic condition
To investigate the functional role of PC in HSKMSCs myogenic differentiation under hypoxic-ischemic condition, we first incubated HSKMSCs in media with saline or different concentrations of PC (5, 10, 15, 20 ug/ml) for 24h. As shown in Fig. 2a, with PC concentration increased above 5 ug/ml, cell status of HSKMSCs got poor, which suggested that PC concentration no more than 5 ug/ml may be suitable induction concentration.
Quiescent satellite cells express the transcription factor Pax7, to evaluate the effect PC on satellite cell at baseline, we treated the cells with saline, 2.5 ug/ml PC or 5 ug/ml PC for 3 and 5 days in the normal condition, and then detected the expression of Pax7 by western blot (Fig. 2b) and immunofluorescence (Fig. 2c). The results showed that there was no significant difference in the expression of Pax7 among three groups.
When SKMSCs were subjected to hypoxic-ischemic condition, cells are activated to induced cell fusion and multinucleated myotubes formation. Cells were treated with saline, 2.5 ug/ml PC or 5 ug/ml PC for 3 and 5 days. The results showed that in the normal condition, there were none myotubes been investigated. While in hypoxic-ischemic condition, myotubes were presented in three groups, but the degree of differentiation was inconsistent: when the differentiated cells been investigated using microscope (Fig. 2d), compared with the saline group, the PC group induced more myotubes, whether the concentration of PC is 2.5 ug/ml or 5 ug/ml. Then, we analyzed the myotubes formation by immunofluorescence analysis (Fig. 2e). Compared with saline, the number of multinucleated myotubes in PC group were significantly increased. Meanwhile, no significantly difference was found between 2.5 ug/ml PC and 5 ug/ml PC. And the number of myotubes at day 5 was more than that at day 3. Moreover, myogenin, a marker indicative myogenic differentiation, was found mainly located in the nucleus, while MHC was mainly located in the cytoplasm (Fig. 2e).
Taken together, these data suggest that PC promote myogenic differentiation of HSKMSCs under hypoxic-ischemic condition.
PC induce the expression of myogenic marker genes both in vivo and in vitro
After being induced by PC in vivo and in vitro, some myogenic differentiation marker proteins are examined. At same time, studies have showed that p38-MAPK signaling pathway may involve in myogenic differentiation (41), so the expression of p38-MAPK and p-p38-MAPK was been investigated. In the ischemia gastrocnemius of mice, Western blot results showed that the protein level of MyoD and p-p38-MAPK in PC-treated were remarkably increased compared to that in H2O-treated, but without an effect on p38-MAPK. (Fig. 3a).
Consistent with the result in vivo, we also got similar results in vitro, two dosage of PC all improved the expression of myogenic related protein, such as MyoD, myogenin and MHC (Fig. 3b). Moreover, the protein expression in 2.5 ug/ml PC was more obvious than that in 5 ug/ml and increased over time. Taken together, we choose 2.5 ug/ml PC treatment in the following study.
To further investigate whether PC also involves oxidative stress in addition to affecting myogenic differentiation, the ROS levels were further detected in HSKMSCs under hypoxic-ischemic condition at days 1, 3 and 5 (Fig. 3c, 3d). The results showed that elevated ROS level was induced by hypoxic-ischemic condition. Compared with saline, two dosage of PC induction all resulted in significantly lower ROS levels. Compared with 2.5 ug/ml PC, 5 ug/ml PC induction resulted in lower ROS levels relatively (Fig. 3c, d).
PC induced the expression of miR-133b to promote myogenic differentiation through p38-MAPK pathway under hypoxic-ischemic condition
To investigate whether miRNAs are involved in PC mediated myogenic differentiation when limbs suffer from ischemic injury. Unilateral limb ischemia mice were treated with 20 mg/kg PC and H2O for 7 days, ischemic gastrocnemius was collected to detect the significant differentially expressed miRNAs using microarray analysis, eight significant differentially expressed genes were analyzed by clustering analysis (Fig. 4a). Combine with the results of the RT-qPCR, the miR-133b-3p of mice was significant elevated and its gene sequence was consistent with the miR-133b of human. And they sharply increased both in vivo and vitro with PC treatment (Fig. 4b).
We further transfected HSKMSCs with miR-133b mimic, inhibitor or negative control, then followed by hypoxic-ischemic treatment for 3 days to determine the protein expression. The results showed that upregulation of miR-133b remarkably improved the expression of MHC, MyoD, myogenin and p-p38 MAPK compared to miR-133b mimic NC. Correspondingly, downregulated miR-133b inhibited the expression of these proteins and partially reversed the effect of PC on these proteins (Fig. 4c, 4d).
We further analyzed the myogenic differentiation in HSKMSCs by immunofluorescence analysis. As shown in Fig. 4e, after transfection with miR-133b mimic, the number of multinucleated myotubes was significantly increased compared with transfection with miR-133b mimic NC. On the contrary, after downregulated miR-133b, the number of multinucleated myotubes was remarkably inhibited.
These results suggested that miR-133b induced by PC, was involved in myogenic differentiation by regulating myogenic associated gene through p38-MAPK pathway.
MiR-133b directly targets MKP1 in HSKMSCs
miRNAs posttranscriptionally inhibit the translation of target genes, to investigate which target genes are involved in miRNAs mediated myogenic differentiation when ischemic limbs treated with PC. Bioinformatic analyses were used to predict miRNA target (Supplementary Fig 2). The result shown that MKP1 may be a candidate target gene for miR-133b, the next experiment was to confirm it. Firstly, the expression of MKP1 were obviously attenuated in vivo and in vitro with PC treatment (Fig. 5a). Then, upregulated miR-133b obviously inhibited the MKP1, as expected, inhibition of MKP1 was indeed reversed by downregulated miR-133b (Fig. 5b). These data suggest that regulation of miR-133b altered MKP1 protein expression in HSKMSCs.
Secondly, As shown in Fig. 5c, silencing of MKP1 facilitated the p-p38-MAPK and promoted the expression of MHC, MyoD, myogenin. We further both silencing of MKP1 and inhibit miR-133b in HSKMSCs, the results showed that silencing of MKP1 had the ability to block the miR-133b inhibitor mediated inactivated myogenic related genes.
To analyzed whether miR-133b directly targets MKP1, As shown in Fig. 5d, the predicted interactions between miR-133b and its target sites in the MKP1 3′-UTR showed that good base pairing with the seed region. We next used Luciferase reporter assay to investigated whether miR-133b directly bind to the 3′-UTR seed sequence of MKP1 mRNA. Compared to NC, the results showed that upregulation of miR-133b remarkably inhibited the luciferase reporter activity. In addition, upregulation of miR-133b suppressed the reporter activity were reversed by the mutations, which eliminated the predicted binding site with MKP1 in the MKP1 3′-UTR.
Briefly, the results suggested that miR-133b negatively regulate MKP1 expression by directly target it.